The erythrocyte sedimentation rate and other markers of inflammation in cats tested for Leishmania infantum and feline immunodeficiency virus antibodies

Background In endemic areas, Leishmania infantum and feline immunodeficiency virus (FIV) co-infection occurs in cats, and may favour a progressive course of feline leishmaniosis. Abnormalities in serum protein fractions have been reported, but inflammation markers have scarcely been studied. Erythrocyte sediment rate (ESR) is a marker of inflammation that is poorly used in veterinary medicine, but it has been evaluated in EDTA blood using a recently introduced automatic device. We studied ESR and a pool of feline markers of inflammation (MoI) in cats L. infantum (Li+) and/or FIV antibody-positive (Li+FIV+/FIV+) with the aims (a) to evaluate ESR as MoI in cats with the infectious and clinical conditions considered and (b) to provide data about a pool of MoI never investigated in the feline infections studied and in other cat diseases before. Methods This prospective controlled study included 35 study group cats (Li+, n = 20; FIV +, n = 8; Li+FIV+, n = 7) and ten healthy antibody-negative control cats. Clinical findings at physical examination and selected clinical pathological abnormalities related to inflammation were statistically analysed in relation to the infectious status and ESR values. Results ESR values were higher in Li+, FIV+, and Li+FIV+ cats compared with control cats, and 40% of the study group cats had ESR values above the reference interval (RI). ESR positively correlated with some positive MoI and negatively with some negative MoI studied. Additionally, a higher prevalence of ESR values above the RI has been detected in cats with hypoalbuminemia or hypergammaglobulinemia and higher ESR values were measured in cats with serum protein electrophoresis (SPE) fraction abnormalities. Correlations were also found with erythrocytes, hemoglobin, hematocrit and some erythrocyte indices. FIV+ and Li+FIV+ cats had a higher prevalence of increased ESR values, and almost all had SPE abnormalities and more severe clinical presentations compared with Li+ cats. Conclusions Abnormal levels of MoI were found in almost all parameters studied, particularly in FIV+ and Li+FIV+ cats. Also, ESR can be used as a marker of inflammation in cats with L. infantum and/or FIV infection. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-024-06396-1.


Background
Leishmaniosis caused by Leishmania infantum is a zoonotic disease affecting mainly dogs in endemic areas of the Mediterranean Basin and the Americas, and dogs are also considered the main domestic reservoirs of the parasite [1].Leishmaniosis is less frequently observed in cats; however, it is considered an emerging feline disease [2].The course of L. infantum infection in dogs is influenced by the host immune response, with progressive infection and development of disease when the parasite induces a marked humoral immune response and downregulates cell-mediated immunity [3,4].Leishmania infantum-specific production of antibodies and IFN-γ have been reported in cats exposed to the parasite in endemic areas [5].Similarities in the immunopathogenesis of L. infantum infection have been observed in a study comparing cats and dogs, but cats showed a lower level of both humoral and cell-mediated immune response [6].Feline immunodeficiency virus (FIV) co-infection may play a role as a risk factor for feline leishmaniosis (FeL), and significant associations between the two pathogens have been reported [7][8][9][10][11][12].
Although dermatological lesions are a frequent reason for veterinary consultation of cats with FeL [13,14], systemic signs and clinical pathological abnormalities are usually detected because the disease is associated with a generalised infection [13].Mild-to-moderate, non-regenerative anaemia and proteinuria are common findings, while other cell blood count (e.g.pancytopenia, thrombocytopenia) and serum biochemical (e.g.hypoalbuminaemia, increased liver enzymes, azotaemia) changes are rarer [13].Complete blood count (CBC) changes are known to occur in inflammatory diseases and in endemic areas, some of these CBC abnormalities were more frequently observed in L. infantum-positive cats and in cats co-infected with FIV [15].Hyperproteinaemia with hyperglobulinaemia and hypergammaglobulinaemia are the most prevalent serum abnormalities [13].Some studies investigated the electrophoretic fractions of serum proteins in cats and reported increases of the α 2 , β 2 and γ-globulins with polyclonal gammopathies in L. infantum-infected cats [16,17].Changes in total proteins and electrophoretic protein fractions are routinely evaluated and monitored in L. infantum-infected dogs, where hypoalbuminaemia, increases of α 2 -globulins and γ-globulins can be observed when progression to disease occurs [18][19][20][21][22].An intense inflammatory response is generally observed in leishmaniotic dogs, and abnormalities in various acute-phase proteins have been reported in their clinical pathological evaluation [19].Serum amyloid A (SAA) is a feline major positive acute phase protein (APP) as the concentration increases rapidly and significantly (10-100 times) in serum in case of proper stimulation, and it also decreases fast [23].SAA is the main feline marker of acute inflammation, and it is usually included in the routine biochemical profile of cats; however, it has been scarcely investigated in cats with FeL.In a recent case of FeL with ulcerative dermatitis and chronic uveitis, increased values of SAA have been reported at diagnosis and changes of concentration have been considered useful for monitoring the course of disease [24].Two studies included SAA in the clinical pathological investigations of cats tested for L. infantum and positive cats did not show higher values compared with healthy control cats.There are no publications evaluating other APPs in cats with L. infantum infection or disease.However, α-1-acid glycoprotein (AGP) and haptoglobin (Hp) are positive moderate APPs (i.e.increasing less rapidly, < tenfold and decreasing slower than major APPs) investigated in various infectious and non-infectious feline diseases [23].In particular, AGP is considered a specific clinical pathological marker of feline infectious peritonitis [23].Albumin and total iron binding capacity (TIBC) concentrations are defined as negative APPs as they decrease in case of inflammation, and they do it slowly [23].Similarly, serum iron is a negative marker of inflammation [25], and it is also reduced in case of iron depletion (e.g. in chronic haemorrhage), but in this latter case, a normal-to-increased value of TIBC is found while in inflammatory conditions TIBC value is normal-todecreased [26].As both causes of low iron levels induce non-regenerative anaemia, the simultaneous evaluation of iron and TIBC contributes to differentiating anaemia of inflammatory disease from anaemia of iron deficiency [26].
In recent years, blood cell ratios (BCRs) (NL, neutrophil-to-lymphocyte; ML, monocyte-to-lymphocyte; PL , platelet-to-lymphocyte ratios) have been investigated as cost-effective and easily accessible diagnostic and prognostic markers of phlogistic and neoplastic [27,28] diseases has been proposed in cats.The relationships between NL, ML and PL ratios and some markers of inflammation routinely measured in cats (SAA, albumin, globulin, albumin-to-globulin ratio and leukocyte alterations suggestive of inflammation) have been investigated, and authors concluded that these BCRs are good markers of inflammation in cats [29].However, no studies have currently evaluated their changes in FeL, FIV and feline leukaemia virus (FeLV) infections or co-infection in cats.
The erythrocyte sedimentation rate (ESR) is an old marker of inflammation, and the test measures in citrated blood the distance (mm/h) that red blood cells (RBC) cover while they sediment inside a vertical graduated test tube.The Westergren assay is the gold standard method for performing ESR measurements in humans and animals.Westergren method has been neglected in veterinary medicine, particularly in cats because of the limitation of dedicating 1 mL of blood to this test [30][31][32].New automatic technologies have been developed in human medicine with the aim to perform ESR in laboratories managing high volume of samples, to speed up the test and to avoid any withdraw of blood from sample tubes for maximum user safety.In some cases, the new technologies validated the use of K 3 EDTA blood instead of citrated blood, and this latter characteristic opened the possibility of measuring ESR with the same EDTA blood tube used for CBC.In 2020, a new automated portable ESR device operating with the K 3 EDTA tubes used for CBC has been marketed for measuring ESR in dogs, cats and horses (MINI-PET, DIESSE Diagnostica Senese, Monteriggioni, Siena, Italia).The instrument has been evaluated in dogs [32][33][34], horses [35] and cats [36,37].The two studies performed in cats investigated the effects of some analytical (test duration) and blood sample (packed cell volume, temperature and length of storage of blood) variables on the ESR values [36] and the changes observed in feline chronic kidney disease [37].Cavalera et al. [34] measured the ESR in dogs with canine leishmaniosis (CanL) with MINIPET and reported increased values in dogs affected by active form of disease compared with exposed or healthy dogs [34].
This prospective study assessed values of ESR and of other markers of inflammation in clinically evaluated cats with antibody titres positive to L. infantum (Li + ), FIV (FIV + ) and both pathogens (Li + FIV + ), compared with healthy antibody-negative cats, and investigated correlations between their values.The aims were: (a) to test the hypothesis that ESR values measured in EDTA blood with an automated point-of-care (POC) device that can be used as a marker of inflammation in cats with the infectious and clinical conditions considered and (b) to provide data about a pool of markers of inflammation never investigated in the feline infections studied and in other cat diseases before.

Study sites, cat enrollment and sampling procedures, and study groups
From March to December 2023, cats that presented for annual health checks, minor medical problems, elective, minor or orthopedic surgeries, referred to the Clinica Veterinaria Camagna-VetPartners (Reggio Calabria, Italy) were prospectively evaluated.The study protocol has been approved by the ethics committee of the Department of Veterinary Sciences of the University of Messina (no.05/23), and the cat owners signed the informed consent form, which provided relevant information about the study protocol.
Cats older than 6 months and irrespective of sex, weight, breed, reproductive and clinical status were evaluated.Exclusion criteria included: age below 6 months, critical clinical conditions, diagnosis of neoplasia, endocrine, allergic and lungworm disease, feline infectious peritonitis, severe acute upper respiratory tract disease, feline panleukopaenia, serum positivity to FeLV-p27 antigen rapid test, and treatments with at least one of the following drugs in the last 30 days: corticosteroids, immunosuppressive and non-steroidal anti-inflammatory drugs.Demographic data (i.e.breed, age and sex), baseline information (i.e.clinical history, concomitant treatments and previous diseases), physical examination findings and any diagnosis of comorbidities were recorded for each cat.A total of 3-5 mL of blood were taken from each cat: 1 mL was placed into a K 3 EDTA tube and used for ESR and CBC evaluations.The remaining blood was used to perform blood smears and to obtain serum after clotting in a plain tube and centrifugation for biochemical, electrophoretic and antibody testing.In the case of cats admitted for elective, minor or orthopaedic surgeries, blood collection was performed before the surgical procedures.Urine samples (about 5 mL) were obtained by cystocentesis or free catch and used for urinalysis and urine protein to creatinine ratio (UPC) evaluations.
The study group included cats with various clinical and/or clinical pathological abnormalities and resulting antibody positive to L. infantum and/or FIV, and negative for FeLV.Indoor, client-owned cats that were clinically healthy according to history, physical examination and clinical pathological evaluation, and antibody negative to L. infantum, FIV and FeLV infections were enrolled as a control group.

Clinical pathological and ancillary investigations
The CBC was performed using the haematology analysers Siemens Advia 2120 (MyLav-La Vallonea, Laboratorio di Analisi Veterinarie s.r.l., Milan, Italy), ProCyte Dx (IDEXX laboratories, Westbrook, ME, USA) or Futurlab DF50 (Padova, Italy).The absolute counts of neutrophils, monocytes and platelets were divided by the absolute count of lymphocytes and NL, ML and PL ratios were calculated [29].The platelet counts were excluded, and the PL ratio was not calculated when platelet clumps were detected in blood smears.
The evaluated parameters' reference intervals (RI) were internally estimated by MyLav-La Vallonea, Laboratorio di Analisi Veterinarie s.r.l., Milan, Italy, and are reported in a Supplementary Table (Table S1) [39].In case of ESR, the RI considered was within values of 2.5th and 97.5th percentile of control group cats (1-48 mm/h).
Due to technical reasons, insufficient volume of serum and the lack of urine samples, not all parameters of SBP, SPE and urinalysis tests were evaluated in all cats, as shown in Table S1.
Routine diagnostic investigations were additionally performed in study group cats to rule out comorbidities based on a problem-oriented medical approach.In particular, fine-needle insertion (FNI) was performed in case of enlarged lymph nodes and stained lymph node smears (May Grünwald-Giemsa stain, Merck KgaA, Darmstadt, Germany) were cytologically evaluated [40].

Statistical analysis
Statistical analysis was performed using Jamovi 2.3.28 statistical software program.
The distribution of continuous variables was evaluated by the Shapiro-Wilk test.Spearman's Rho test was used to evaluate the correlation between ESR and the other markers of inflammation.The strength of this relationship, according to the correlation coefficient absolute value (r s ), was qualified as follows: r s = 1: perfect correlation; 1 > r s ≥ 0.8: strong correlation; 0.8 > r s ≥ 0.4: moderate correlation; 0.4 > r s > 0: weak correlation; and r s = 0: no correlation [43].The critical value of r s was established on the basis of the number of pairs of scores for each pair of parameters evaluated [44].Mann-Whitney U test was used to compare age between the control and study groups and to compare the values of ESR and other markers of inflammation between (a) control and study groups, (b) control group and Li + cats, (c) control group and FIV + cats, and (d) control group and Li + FIV + cats.Fisher's exact test was used to compare differences in sex between the study group and control group and to evaluate the association between serological status and clinical signs and parameters with outlier values.Differences were considered significant if P values were < 0.05.

Cats studied and their serological, clinical and clinical pathological evaluations
A cohort of 45 cats was enrolled, and 35 cats were included in the study group, while 10 cats formed the control group.In the study group of cats (21 males and 14 females; mean age 54.5 ± 49.6 months), 17 cats were indoors, 15 were outdoors, and three cats lived both indoors and outdoors.Control group cats (seven females and three males; mean age 26 ± 35.3 months) were all indoors.No significant differences were found between the two groups concerning age and sex.
Abnormalities were observed at physical examination (65.7%; 23/35) and the clinical pathological evaluation (94.3%; 33/35) of the study group cats, and they are described as individual cats with their antibody-positive status in Table 1.Various oral disorders (periodontitis, n = 9; stomatitis, n = 5; dental calculi, n = 4; tongue ulcers, n = 1) were found.Enlarged lymph nodes (n = 11) was the more prevalent clinical sign and lymphoid hyperplasia was cytologically diagnosed.Crusty dermatitis (n = 2, with alopecia and pruritus in one case), alopecia (n = 2) and squamous dermatitis with alopecia (n = 1) were the dermatological lesions detected.Conjunctivitis (n = 3) and keratoconjunctivitis (n = 1) were the ophthalmic findings observed.Rhinitis (n = 1) and diarrhoea (n = 1) affected one single cat each.Flea infestation was detected in one cat, but it was not associated with clinical manifestations.

Values of ESR and data analysis of the markers of inflammation
Descriptive statistics of values of ESR and the other markers of inflammation considered are reported in Table 2.

Comparisons between L. infantum-positive and control group cats
Outlier values of L. infantum antibody-positive cats were not statistically different from the values of the control group cat.However, values of ESR (P = 0.031), leucocytes (P = 0.013), neutrophils (P = 0.011) and haptoglobin (P = 0.019) were significantly higher compared with the control group (Table 2).Although SAA concentrations resulted in significantly higher (P = 0.013) values in the control group, all values were within the RI in both groups (Table 2).

Discussion
This study showed that cats antibody-positive to L. infantum and/or FIV have higher ESR values than healthy antibody-negative cats, and 40% of them have values above the RI obtained from control group cats.We investigated ESR in cats infected by L infantum and FIV because in endemic areas cats with FIV have a higher risk for L. infantum infection, and in turn the coinfection is a risk factor for the development of FeL [7,13].Feline leishmaniosis and FIV infections both have a chronic course with clinical pathological changes reflecting chronic inflammation [13,45].The ESR is an inflammatory index due to the fact that the sedimentation of erythrocytes suspended in their plasma is faster in case of inflammatory conditions [33].Dysprotidaemia associated with inflammation is one causative factor for the increased speed of RBC aggregation that precedes their sedimentation, and the physical mechanism of this process is still debated [46].Hypoalbuminaemia, increases of positive APPs and immunoglobulins (IgM and IgG) occur together in inflammatory conditions, and contribute to elevate the speed of ESR [47].This pathomechanism explains both the positive and negative correlations ESR, erythrocyte sedimentation rate; MCHC, mean corpuscular hemoglobin concentration; A/G, albumin-to-globulin ratio; NL, neutrophil-to-lymphocyte ratio; ML, monocyte-to-lymphocyte ratio; PL, platelet-to-lymphocyte ratio; IgG, immunoglobulins G; IgM, immunoglobulins M; Fe, iron; TIBC, total iron binding capacity; AGP, α-1 acid glycoprotein; * significant difference compared with the control group found between ESR and negative (albumin, albumin-toglobulin ratio and the TIBC) and positive (total globulins, γ-globulins, IgG, IgM, α 1 -globulins and haptoglobin) markers, respectively.For the same reason, a higher prevalence of ESR values above the RI has been detected in cats with hypoalbuminaemia or hypergammaglobulinaemia, and cats with hypoalbuminaemia, hyperglobulinaemia, low A/G, and increased α 2 -globulins and γ-globulins had significantly higher ESR measures.Changes in these parameters are influenced by inflammation, which in turn contributes to increasing the speed of ESR.An additional cause for the increased speed of ESR is the reduction of Hct as it changes the proportion among RBC and plasma.As expected, Hct, RBC and Hgb values were negatively correlated with ESR measures.Moreover, a positive correlation with some erythrocyte indices (MCV and MHC) were observed as seen in humans [47,48].Mild to moderate anaemia occurs in chronic inflammation, and it is reported in L. infantum-infected cats, while severe anaemia is more frequent in FIV-positive cats [15].Three cats had mild (3L, 4LF) or moderate (4F) normocytic normochromic non-regenerative anaemia, and they had multiple abnormalities in markers of inflammation, including increased ESR values in two of them.
Interestingly, a higher prevalence of increased ESR values was found in the FIV + cats and in those with dual infection.The Li + cats had low antibody titres (≤ 2-fold dilutions above the cut-off ) and a lower prevalence of clinical signs compared with Li + and Li + FIV + group.Therefore, they may be in an early stage of the infection or be animals with a non-progressive form of infection.Longitudinal studies are needed to understand the dynamics of L. infantum infection in cats and markers of inflammation-including ESR-are useful to know for supporting prognostic evaluations.Based on the physical examination and CBC findings, the course of FIV infection was not advanced in studied cats as none of them suffered from myelosuppression, opportunistic infections or neoplasia [49]; however, their health status was in general more compromised compared with cats of Li + group when evaluated at physical examination (Table 1).Clinical findings observed in the study group were manifestations quite commonly reported in cats with oral diseases (34.2%; 12/35) and with enlarged lymph nodes (31.4%; 11/35) being the more prevalent, similar to a previous study in cats from the same area where outdoor cats had higher prevalences of oral disease and enlarged lymph nodes compared with indoors [50].However, lymph node enlargement was significantly more prevalent in FIV + and FIV + Li + cats.Donato et al. [50] investigated clinical epidemiological findings in cats studied for feline morbillivirus and the co-infection with FIV [51].They found lymph node enlargement in about 30% of cats, but it was significantly correlated with the co-infection with FIV [47].These previous field data and the present findings are explained by observations from a long-term experimental study that evidenced lymphoid hyperplasia more than five years after the experimental infection in monitored cats [52].Before the terminal phase of FIV infection, most cats have a slow and progressive decrease in CD4 + T lymphocytes and show a dysregulation of immune response with chronic infections and polyclonal abnormal B-cell activation, both causing hyperglobulinaemia and hypergammaglobulinaemia also due to autoantibodies production [52][53][54].
In this study almost all FIV + and Li + FIV + cats (14/15) had abnormalities at the SPE evaluation and in a significantly higher number of them increased γ-globulins (both groups) and α 1 -globulins (FIV + group) levels were observed.The prevalence of ESR outliers was higher in FIV + (62.5%) and Li + FIV + (42.8%) with respect to Li + cats (30%).The difference was not significant but importantly this finding is in line with those from α-and γ-globulins.An accurate evaluation of acute phase reaction in routine clinical pathological evaluation of cats is based on an APP profile which includes at least one positive major (SAA) and one positive moderate (Hp) APP [55].Apart from SAA, Hp, and albumin as a negative APP routinely added in the SBP, we correlated ESR measures with many other blood parameters influenced by inflammation because they may be involved through different pathomechanisms causing their upregulation or depletion from blood, have different biological properties and kinetics, not completely known in cats [23].We found increased values of at least one of the positive (SAA, AGP and Hp) and negative (albumin, TIBC, and Fe) AP markers studied in overall 15 cats.Six of them had from two to four abnormal AP marker values, and they were FIV + (n = 3) and Li + FIV + (n = 3) cats, confirming that a more complex and probably multifactorial pathomechanism of inflammation may exist in FIV infection and co-infection with L. infantum.The AGP is an α 1 -globulin, but it was found to be elevated (912 μg/mL) in only one studied cat (3L), which had no signs at physical examination but mild non-regenerative anaemia, increased γ-globulins (23.7 g/L) and ESR (55 mm/h) values.No cats had abnormal TIBC values, including cats with low concentrations of Fe (n = 8) or albumin (n = 4).However, this parameter can be unfortunately found within reference range both in cases of iron depletion and sequestration [26].Among the positive APPs studied, only haptoglobin showed a moderate correlation with ESR, with increased values in five cats (18L, 2F, 5F, 2LF, 5LF).Haptoglobin is an α 2globulin synthesised in the liver and well known for binding free Hgb, which is a by-product of hemolysis with pro-inflammatory activity [56].However, increased α 2globulin values were seen only in cats 5F and 5LF.Immunoglobulins M and G were higher in FIV + (IgG) and Li + FIV + (both IgM and IgG) cats compared with control cats, and values above RI were found only in cats with dual infections (LF1, LF5, LF6).
Uva et al. [37] found in cats with chronic kidney disease (CKD) increased values of both ESR and SAA compared with the healthy control cats, but they did not find the correlation between the two parameters [34].In fact, they reported that ESR was significantly elevated in more advanced International renal interest society (IRIS) stages of CKD (IRIS stage 3 and 4) compared with SAA.In this study, six cats had CKD (Table 1) with four IRIS stage 1 cats (9L, 11L, 5F, 4LF), one stage 2 (4F) and one stage 3 (5LF) cats diagnosed.Elevated ESR values were measured in cats 9L, 4F, 5F and 5LF, while increased concentrations of SAA were only in cats 5F and 4LF.The presence of FIV and/or L. infantum infections in these cats and the limitations due to a small number of observations do not allow to speculate about the role of CKD in these abnormalities.Moreover, the ESR values (median = 13.5;25th-75th percentile = 9.75-24 mm/h) of healthy control cats were lower (13.5 mm/h) in this study compared with Uva et al. [37] (median = 30.0,25th-75th percentile = 16.0-37.0mm/h) [34].Both control groups included only ten adult individuals, which is a limitation.Extensive studies considering demographic variables are required to assess RI of ESR.Apart the study of Uva et al. [34,37], no other data are available about ESR values in cat diseases and, despite the limitations from the small number of cats studied, we think that data obtained in the present study demonstrated that ESR values measured in EDTA blood with a POC device can be used as marker of inflammation in cats with the infectious and clinical conditions considered.Investigations have to be extended to obtain more robust results and perform multivariate analyses.There is a great need to have reliable, automated and easily accessible POC devices for clinical pathological investigations, and even more so to obtain these data with the smallest volume of blood collected from cats.

Conclusions
This study found that ESR measured in EDTA blood with an automated device is a marker of inflammation in cats with L. infantum and/or FIV infections.Cats with FIV or dual infections have a more severe inflammatory condition, as shown by results from a pool of markers of inflammation never simultaneously assessed in the feline infections studied and in other cat diseases.

Table 1
Clinical and clinical pathological abnormalities and ESR values in the study group cats based on their antibody positivity to L. infantum and/or FIV NTR ↓ Fe, ↑ SAA ↑ Hp ↓ albumin ↑ globulins ↑ γ-globulins ↓ A/G ↑ NTR clinical pathological abnormality was the increase of γ-globulins (n = 18) and polyclonal gammopathies were observed.Increases of α 1 (n = 9) and α-2 (n = 2) globulins were seen and associated with polyclonal gammopathy, respectively, in five and one cats.A low value of Fe was found in nine cats, and two out of the three cats with anaemia (normocytic normochromic, non-regenerative; two mild and one moderate) had low Fe concentrations (4F, 4LF), as well as the unique cat with thrombocytosis (15L).Low A/G values were detected in five cats, four of which had hypoalbuminaemia, and this was associated with increased concentrations of Hp, SAA or IgM or low Fe measures.

Table 3
Correlation matrix between erythrocyte sedimentation rate values and the markers of inflammation considered in the cohort of 45 cats